Eosinophils are bi- or multi-nucleate white blood cells which contain basophilic or eosinophilic granules formed during their development by highly active golgi and ribosomal machinery. The plasma membrane is not structurally distinct from that of other leukocytes, but it is characterized by immunoglobulin (Ig) receptors, particularly IgG and IgE. These cells are formed throughout life from pluripotent stem cells and play a crucial role in systemic defense protecting the body from microorganisms and foreign proteins. In comparison to a total of 7000 white blood cells per microliter of blood, the number of eosinophils is normally about 160 cells per microliter. Eosinophils, generally six days worth, are formed and stored in the bone marrow until they are recruited to the site of inflammation or invasion.
Eosinophils have a special function in parasitic infections. They attach to parasitic larvae, presumably via their Ig receptors, and undergo degranulation in response to interleukin-5 (IL-5), IL-3, granulocyte/monocyte cell stimulating factor (GM-CSF) produced by activated T cells and mast cells of the host (Abu-Ghazaleh R I, Kita H, Gleich G J (1992) Immunol Ser 57:137-67) or other factors produced by the parasite. Degranulation releases many active species including the following: 1) hydrolytic enzymes such as peroxidase, acid phosphatase, phospholipase, B glucuronidase, ribonuclease, arylsulfatase and cathepsin; 2) highly reactive superoxides; and 3) major basic protein (MBP), an arginine-rich potent larvicidal polypeptide and eosinophil cationic protein (cf. Capron M (1992) Mem Inst Oswaldo Cruz 87(S5):83-9). Eosinophils are produced in great quantities in persons with helminthic infections such as hookworm, schistosomiasis, toxocariasis, trichuriasis, filariasis, strongyloidiasis, echinococcosis, cysticercosis, and trichinosis, etc.
Large numbers of eosinophils also collect in tissues such as the heart, lungs, central nervous system, sinuses and skin where allergic reactions commonly occur. They are chemoattracted to the site of inflammation or invasion by eosinophil chemotactic factor, platelet activation factor, complement 5a, or IL-5 which are released by mast cells and basophils during the allergic reaction. Eosinophils neutralize slow reacting substance of anaphylaxis (a mixture of leukotrienes) and histamine released by mast cells and basophils, produce eosinophil derived inhibitor which prevents degranulation of mast cells, and phagocytize antigen-antibody complexes--all of which downregulate the hypersensitivity response.
Eosinophilia, an excess of eosinophils--more than 500 per microliter of blood--is commonly observed in patients with allergies, hay fever, asthma and reactions to drugs as common as aspirin, sulfonamides and penicillins. Eosinophilia is also associated with rheumatoid arthritis and cancers such as Hodgkins lymphoma, chronic myelogenous leukemia, and carcinomas of the lung, stomach, pancreas, ovaries, uterus and liver. Eosinophilia may cause tissue damage by excessive degranulation and is usually treated with glucocorticoid chemotherapy.
Eosinophils, their morphology, function and relation to disease are reviewed, inter alia, in Guyton AC (1991) Textbook of Medical Physiology, W B Saunders Co, Philadelphia Pa.; Isselbacher K J et al (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York City, pp. 1437-1504; and Zucker-Franklin D et al (1988) Atlas of Blood Cells, Function and Pathology, Lea and Febiger, Philadelphia Pa.
As is the case in inflammation of other tissues, leukocytes including monocytes, macrophages, basophils, and eosinophils infiltrate the inflamed area of the pancreas. Their primary role is to clean up the site of the inflammation; however, macrophages may produce powerful oxidants and proteases which contribute to tissue destruction. Leukocytes also secrete a range of cytokines which recruit other cells to the area.
The investigation of the critical, regulatory processes by which white cells proceed to their appropriate destination and interact with other cells is underway. The current model of leukocyte movement or trafficking from the blood to injured or inflamed tissues comprises the following steps. The first step is the rolling adhesion of the leukocyte along the endothelial cells of the blood vessel wall. This movement is mediated by transient interactions between selectins and their ligands. A second step involves cell activation which promotes a more stable leukocyte-endothelial cell interaction mediated by the integrins and their ligands. This stronger, more stable adhesion precipitates the final steps--leukocyte diapedesis and extravasation into the tissues.
The chemokine family of polypeptide cytokines possesses the cellular specificity required to explain leukocyte trafficking in different abnormal, inflammatory or diseased situations. First, chemokines mediate the expression of particular adhesion molecules on endothelial cells; and second, they generate gradients of chemoattractant factors which activate specific cell types. In addition, the chemokines stimulate the proliferation of specific cell types and regulate the activation of cells which bear specific receptors. These activities demonstrate a high degree of target cell specificity.
The chemokines are small polypeptides, generally about 70-100 amino acids (aa) in length, 8-11 kD in molecular weight and active over a 1-100 ng/ml concentration range. Initially, they were isolated and purified from inflamed tissues and characterized relative to their bioactivity. More recently, chemokines have been discovered through molecular cloning techniques and characterized by structural as well as functional analysis.
The chemokines are related through a four-cysteine motif which is based primarily on the spacing of the first two cysteine residues in the mature molecule. Currently the chemokines are assigned to one of two families, the C--C chemokines (.alpha.) and the C--X--C chemokines (.beta.). Although exceptions exist, the C--X--C chemokines generally activate neutrophils and fibroblasts while the C--C chemokines act on a more diverse group of target cells which include monocytes/macrophages, basophils, eosinophils, T lymphocytes and others. The known chemokines of both families are synthesized by many diverse cell types as reviewed in Thomson A. (1994) The Cytokine Handbook, 2d Ed. Academic Press, NY. The two groups of chemokines will be described in turn.
At this time, relatively few C--C chemokines have been described, and they appear to have less N-terminal processing than the C--X--C chemokines. A brief description of the known human (and/or murine) C--C chemokines follows. The macrophage inflammatory proteins alpha and beta (MIP-1.alpha. and .beta.) were first purified from stimulated mouse macrophage cell line and elicited an inflammatory response when injected into normal tissues. At least three distinct and non-allelic genes encode human MIP-1.alpha. and seven distinct genes encode MIP-1.beta..
MIP-1.alpha. and MIP-1.beta. consist of 68-69 aa which are about 70% identical in their acidic, mature secreted forms. They are both expressed in stimulated T cells, B cells and monocytes in response to mitogens, anti-CD3 and endotoxin, and both polypeptides bind heparin. While both molecules stimulate monocytes, MIP-1.alpha. chemoattracts the CD-8 subset of T lymphocytes and eosinophils, while MIP-1.beta. chemoattracts the CD-4 subset of T lymphocytes. In mouse, these proteins are known to stimulate myelopoiesis.
I-309 was cloned from a human .gamma.-.delta.T cell line and shows 42% aa identity to T cell activation gene 3 (TCA3) cloned from mouse. There is considerable nucleotide homology between the 5' flanking regions of these two proteins, and they share an extra pair of cysteine residues not found in other chemokines. Such similarities suggest I-309 and TCA3 are species homologs which have diverged over time in both sequence and function.
RANTES is another C--C chemokine which is expressed in T cells (but not B cells), in platelets, in some tumor cell lines, and in rheumatoid synovial fibroblasts. In the latter, it is regulated by interleukins-1 and -4, transforming nerve factor and interferon-.gamma.. The cDNA cloned from T cells encodes a basic 8 kD protein which lacks N-linked glycosylation and is able to affect lymphocytes, monocytes, basophils and eosinophils. The expression of RANTES mRNA is substantially reduced following T cell stimulation.
Monocyte chemotactic protein (MCP-1) is a 76 aa protein which appears to be expressed in almost all cells and tissues upon stimulation by a variety of agents. The targets of MCP-1, however, are limited to monocytes and basophils in which it induces a MCP-1 receptor:G protein-linked calcium flux (Charo I, personal communication). Two other related proteins (MCP-2 and MCP-3) were purified from a human osteosarcoma cell line. MCP-2 and MCP-3 have 62% and 73% aa identity, respectively, with MCP-1 and share its chemoattractant specificity for monocytes.
Current techniques for diagnosis of abnormalities in the inflamed or diseased tissues mainly rely on observation of clinical symptoms or serological analyses of body tissues or fluids for hormones, polypeptides or various metabolites. Patients often manifest no clinical symptoms at early stages of disease or tumor development. Furthermore, serological analyses do not always differentiate between invasive diseases and genetic syndromes which have overlapping or very similar ranges. Thus, development of newdiagnostic techniques comprising small molecules such as the expressed chemokines are important to provide for early and accurate diagnoses, to give a better understanding of pathology at the molecular level. Current methods of treating eosinophil-related conditions involve administration of steroids and other drugs with multiple side effects. A new chemokine can be used to develop more specific drugs with fewer side effects.
The chemokine molecules are reviewed in Schall T J (1994) Chemotactic Cytokines: Targets for Therapeutic Development. International Business Communications, Southborough Mass., pp 180-270; and in Paul W E (1993) Fundamental Immunology, Raven Press, New York City, pp 822-826.